Copolymers with varying ratios of muscle and Acanthamoeba actin were prepared and the interaction between actin copolymers and heavy meromyosin (HMM) was examined in the presence and absence of tropomyosin (TM). It was found that each type of actin monomer in the copolymers reacted with HMM independent of its neighboring actin monomers. This independence persisted even when all actin monomers in the copolymers bound TM. These results indicate that TM does not affect the actin-myosin interaction by simple physical movement, as proposed by Huxley. Rather, two adjacent subsites on TM can have different effect on the actin monomers they bind. Huxley's model suggests that TM should exert a uniform effect on all bound actins. Even though each subsite of TM can affect actin independently, the binding of TM to actin exhibits a high degree of positive cooperativity. In the presence of Mg2 ion, this cooperativity was abolished when actin filaments were crosslinked with glutaraldehyde. Crosslinked actin, which interacts normally with HMM, fails to bind TM. Crosslinking thus "freezes" actin in a conformation unable to interact with TM. The freedom of actin monomers in the actin filament may be essential for their interaction with TM.